Vision loss is among the top ten disabilities in the United States, which results in a heavy financial burden on society. To remedy this significant problem, the National Eye Institute recently announced the Audacious Goal to regenerate neurons and neural connections in the eye and visual system (://www.nei.nih.gov/audacious/). To accomplish this Audacious Goal, it is necessary to identify the molecular signals needed to activate latent endogenous cells to replace lost host neurons. To identify these potential regulators, we are studying zebrafish, where retinal damage stimulates Mller glia to proliferate and produce neuronal progenitors that regenerate the missing zebrafish neurons. While the human retina also possesses Mller glia, they are unable to regenerate retinal neurons. Identifying the molecular switches that induce the zebrafish Mller glia to initiate the regeneration response may reveal approaches to induce a similar retinal regeneration response in humans. We recently identified tumor necrosis factor alpha (TNFa) and Notch signaling as positive and negative regulators of Mller glia proliferation, respectively. However, the signalin pathways of TNFa and Notch are less clear. Elucidating these pathways could significantly advance the NEI's Audacious Goal by yielding a strategy to regenerate retinal neurons in individuals who suffer from a variety of forms of blindness. Our long-term goal is to identify and characterize the molecular and cellular events required to regenerate the damaged zebrafish retina. We recently found that tumor necrosis factor-alpha (TNFa) is produced in the dying zebrafish photoreceptors and is necessary and sufficient for Mller glia proliferation. We also observed that repressing Notch signaling is sufficient to induce Mller glia to reenter the cell cycle, suggesting that Notch is a negative regulator of initiating the regeneration response. Our central hypothesis is that dying photoreceptors produce TNFa, which binds receptors on the Mller glia and activates Stat3. Stat3 then regulates the expression of Ascl1a to induce Mller glia proliferation. Additionally, retinal damage represses Notch signaling to increase expression of Ascl1a and Mller glia proliferation, likely through the decreased expression of his/her genes. Aim 1 will explore the components of the TNFa signaling pathway that initiates Mller glia proliferation and in what cells they act, including the ability of TNFa to induce expression of Stat3 and Ascl1a, the potential role of Stat3 in inducing Ascl1a expression, if Stat3 must be activated in Mller glia or another retinal cell type for Mller glia proliferation, and if TNFa activates Stat3 directly or through an intermediate such as NF-?B (NF-kappaB), JNK, or p38. Aim 2 will examine the role of the Notch signaling pathway to maintain Mller glia in a quiescent (non-proliferating) state in undamaged retinas. We will determine if Notch activity must be in the Mller glia to maintain quiescence and determine the identity of the Notch receptor and ligand that are required to keep the Mller glia from reentering the cell cycle in undamaged retinas. Thus, the expected outcomes of this project will reveal the relationships of TNFa and Notch as positive and negative regulators of Mller glia proliferation and how Stat3 and Ascl1a are regulated in the damaged zebrafish retina. We anticipate that the impact of this work will lead to a better understanding of what regulates Mller glia reentry into the cell cycle in the damaged retina. This work will also assist in the development of potential therapeutic approaches that use endogenous Mller glia to regenerate lost retinal neurons in individuals suffering from vision loss.